advances and prospects in forage systems biology and molecular...

Advances and Prospects in Forage

Systems Biology and Molecular Breeding

German Spangenberg

2

Systems Biology: from Genome to Phenome

3

Systems Biology for Transformational

Through-Value Chain Impact

Forage yield

Forage quality

Forage persistence

Biotic stress tolerance

Abiotic stress tolerance

Feed efficiency

Milk composition

Methane

Core genetic traits

Milk composition Products

Health

Nutrition

Plant

symbiome Rumen

microbiome Animal

symbiome Milk

biome

Systems Biology of Forage Grass

Symbiomes and Microbiomes

N H

O

O

H O O O

O

H O

H

C h e m i c a l F o r m u l a : C 3 9 H 5 1 N O 7 E x a c t M a s s : 6 4 5 . 3 6 6 5 5

N H

N H

H N

O

N O

N H O H

O

O C h e m i c a l F o r m u l a : C 2 9 H 3 5 N 5 O 5

E x a c t M a s s : 5 3 3 . 2 6 3 8 2

N N

O

N

N H 2

H 2 N

C h e m i c a l F o r m u l a : C 1 2 H 1 7 N 5 O E x a c t M a s s : 2 4 7 . 1 4 3 3 1 Janthitrem I

ergovaline

peramine

[M+H] + 248.15022

[M+H] + 646.37238

[M+H] + 534.27002 N H

O

H O

O

O

H

O

H O

O H

H


[M+H] + 686.40369

Lolitrem B

N H

O

O

H O O O

O

H O

H


N H

N H

H N

O

N O

N H O H

O

O C h e m i c a l F o r m u l a : C 2 9 H 3 5 N 5 O 5

E x a c t M a s s : 5 3 3 . 2 6 3 8 2

N N

O

N

N H 2

H 2 N

C h e m i c a l F o r m u l a : C 1 2 H 1 7 N 5 O E x a c t M a s s : 2 4 7 . 1 4 3 3 1 Janthitrem I

ergovaline

peramine

[M+H] + 248.15022

[M+H] + 646.37238

[M+H] + 534.27002 N H

O

H O

O

O

H

O

H O

O H

H


[M+H] + 686.40369

Lolitrem B

5

• Asexual filamentous fungi (phylum Ascomycota, family Clavicipitaceae) that form mutualistic symbioses with temperate grasses (subfamily Pooideae)

• Seed transmissible

• Protect host grasses from biotic (e.g. insects and vertebrate herbivores) and abiotic (e.g. drought) stresses

• Produce several bioactive secondary metabolites in planta

• Evolved from sexual grass choke Epichloë pathogens

Neotyphodium spp. Endophytes

E. festucae

Loss of

sexual state

N. lolii

(c. 29 + 4 Mb)

N. lolii x E. typhina

Interspecific

hybridisation

N. sp. LpTG-2

(c. 55 + 6 Mb)

6

From Endophyte Discovery to Pangenome

Analysis Exploiting Global Genetic Diversity – Endophytes

from Perennial Ryegrass

Genetically similar endophytes have a similar toxin profile and origin

Endophytes with reduced toxicity effects are genetically divergent

from the main group

Selection of novel candidate endophytes based on:

DNA profiles

Geographic origin

Toxin profiles

Endophytes cluster into groups based

on geographical origin and toxin

production

Ability to predict likely toxin production

based on genotypic profile Genetic similarity

0.12 0.34 0.56 0.78 1.00

Genetic similarity

0.12 0.34 0.56 0.78 1.00

Middle East

Eastern Europe

Northern Europe

Lolitrem B

Peramine

Middle East

Mediterranean

Western Europe

New World

Lolitrem B

Ergovaline

Peramine

Mediterranean

Western Europe

Eastern Europe

Ergovaline

Peramine

Peramine

LpTG-2

N. lolii

LpTG-3

A broadly-applicable approach for discovery of novel endophytes

Janthitrem

7

In vitro cultures of candidate endophytes

Endophyte genotypes confirmation

Long-term cryopreservation of endophyte cultures

Species No. Isolates Examples

N. lolii 70 ST, NEA2, NEA3, NEA5, NEA6, NEA10, 42 novel endophytes

N. coenophialum 43 E34, E6, 22 novel endophytes

LpTG-2 7 NEA4, NEA11, 3 novel endophytes

LpTG-3 5 NEA12, E1

FaTG-2 4 8907 and 3 novel endophytes

FaTG-3 6 NEA21, NEA23

N. uncinatum 1 E81

Total 136

Discovering Genetically Novel Endophytes

A broad-based, germplasm collection of novel, genetically diverse endophytes 7

8

E9

G4

ST

C9

NA6

Lp19

AR1

NEA3

Genetic similarity

0.12 0.34 0.56 0.78 1.00

Genetic similarity

0.12 0.34 0.56 0.78 1.00

Middle East

Eastern Europe

Northern Europe

Lolitrem

Peramine

Middle East

Mediterranean

Western Europe

New World

Lolitrem

Ergovaline

Peramine

Mediterranean

Western Europe

Eastern Europe

Ergovaline

Peramine

Peramine

NEA12, 15310,15311

E1

Ef E2368

N. lolii

LpTG-3

NEA10

15335

15441

NEA2

15714

NEA6

15931 F2

A1

NEA11

NEA4

LpTG-2

Over 80 ryegrass endophyte strains sequenced

16 N. lolii 3 LpTG-2 4 LpTG-3

Reference genome construction - ST

Representatives of global diversity of perennial ryegrass endophytes

Current commercial endophytes [e.g. AR1, NEA2, NEA3 and NEA4]

New endophytes in pre-commercial development [e.g. NEA10, NEA11, NEA12]

Within cluster analysis of genetic diversity - Endophytes from distinct geographical origins [e.g. ST (Grasslands Samson) – NA6 (Morocco) and C9 (Spain)]

- Endophytes from the same geographical origin [e.g. NEA12 (France) – 15310 and 15311]

Pangenome Analysis of Endophytes

Pangenome analysis across spectrum of genetic, geographic

and taxonomic diversity of endophytes from perennial ryegrass 8

9 Gene present Gene absent Gene partially present

Pangenome Analysis of Endophytes Sequence Diversity in Alkaloid Production Genes

Identification of core and flexible genomes in Neotyphodium endophytes 9

10

Establishing Symbiota in Isogenic Hosts

Developing Diverse Perennial Ryegrass Isogenic Host Panel

Host cultivar Characteristics Number of

TCR genotypesa

TCR genotype used for

inoculation

Tolosa Distinct forage type 1 Tol 03

Bronsyn Standard forage type with robust

endophyte performance 3 Bro 08

Impact Late flowering, dense tillering forage type 3 Imp 04

Meridian Early flowering forage type 1 Mer 05

Barsandra Turf type 1 San 02

Bealey Tetraploid forage type 2 Bea 02

Barsintra Tetraploid forage type 4 Sin 04

Barfest Intergeneric hybrid between Lolium

species parents 3 Fest 02

Materials for symbiome analysis to dissect

endophyte and grass host effects 10

11

Establishing Symbiota in Isogenic Hosts Inoculating Novel Endophytes into Perennial Ryegrass Isogenic Host Panel

Establishing defined symbiota to study Gp x Ge effects 11

12

Endophyte Transcriptome in Symbiota

Perennial ryegrass symbiota; isogenic background; with/without ST endophyte

6 growth conditions: complete media; Low NO3, Low NH4, Low K, Low PO4 and Low Ca

RNAseq libraries; shoots and roots; sequence reads mapped using BLASTn; plant and endophyte transcripts

Endophyte genic sequence reads only observed in tillers of symbiota

Endophyte transcriptome only in symbiotum shoots

genes

0

50000

100000

150000

200000

250000

300000

350000

Full Ca K NH 4

NO 3

PO 4

Full Ca K NH 4

NO 3

PO 4

Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST

Leaves Roots

Reads mapped to endophyte genes with an overlap >40 bp and a percent identity of greater >98 genes

0

50000

100000

150000

200000

250000

300000

350000

Full Ca K NH 4

NO 3

PO 4

Full Ca K NH 4

NO 3

PO 4

Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST Free ST

Shoots Roots

Reads mapped to endophyte genes with an overlap >40 bp and a percent identity of greater >98

13

0

10,000,000

20,000,000

30,000,000

40,000,000

50,000,000

60,000,000

LpL_FRFULL1_80_40

LpL_FRCa1_80_40

LpL_FRK1_80_40

LpL_FRNH1_80_40

LpL_FRNO1_80_40

LpL_FRPO1_80_40

LpL_STFULL1_80_40

LpL_STCa1_80_40

LpL_STK1_80_40

LpL_STNH1_80_40

LpL_STNO1_80_40

LpL_STPO1_80_40

mito

cp

rRNA

gene_sum

Number of reads mapping to plant genes from leaf libraries

0

10,000,000

20,000,000

30,000,000

40,000,000

50,000,000

60,000,000

LpL_FRFULL1_80_40

LpL_FRCa1_80_40

LpL_FRK1_80_40

LpL_FRNH1_80_40

LpL_FRNO1_80_40

LpL_FRPO1_80_40

LpL_STFULL1_80_40

LpL_STCa1_80_40

LpL_STK1_80_40

LpL_STNH1_80_40

LpL_STNO1_80_40

LpL_STPO1_80_40

mito

cp

rRNA

gene_sum


Plant Transcriptome in Symbiota

0

10,000,000

20,000,000

30,000,000

40,000,000

50,000,000

60,000,000

LpL_FRFULL1_80_40

LpL_FRCa1_80_40

LpL_FRK1_80_40

LpL_FRNH1_80_40

LpL_FRNO1_80_40

LpL_FRPO1_80_40

LpL_STFULL1_80_40

LpL_STCa1_80_40

LpL_STK1_80_40

LpL_STNH1_80_40

LpL_STNO1_80_40

LpL_STPO1_80_40

mito

cp

rRNA

gene_sum


0

10,000,000

20,000,000

30,000,000

40,000,000

50,000,000

60,000,000

LpL_FRFULL1_80_40

LpL_FRCa1_80_40

LpL_FRK1_80_40

LpL_FRNH1_80_40

LpL_FRNO1_80_40

LpL_FRPO1_80_40

LpL_STFULL1_80_40

LpL_STCa1_80_40

LpL_STK1_80_40

LpL_STNH1_80_40

LpL_STNO1_80_40

LpL_STPO1_80_40

mito

cp

rRNA

gene_sum


Number of sequence reads mapped to plant sequences in shoot libraries (2.4 to 20.8 million reads per library)

Counts mapping to genes used to identify endophyte-induced or repressed plant genes in symbiota shoots

Of 918 endophyte-regulated plant genes 68 are differentially regulated in shoots

(51 induced and 15 repressed)

Shoot Transcriptome: Endophyte-Regulated Plant Genes

13

14

Plant Transcriptome in Symbiota Root Transcriptome: Endophyte-Regulated Plant Genes

Number of sequence reads mapped to plant sequences in root libraries (2.4 to 20.8 million reads per library)

Counts mapping to genes used to identify endophyte-induced or repressed plant genes in symbiota roots

0

5,000,000

10,000,000

15,000,000

20,000,000

25,000,000

30,000,000

LpR_FR

FULL1_80_40

LpR_FR

Ca1_80_40

LpR_FR

K1_80_40

LpR_FR

NH

1_80_40

LpR_FR

NO

1_80_40

LpR_FR

PO1_80_40

LpR_S

TFULL1_80_40

LpR_S

TCa1_80_40

LpR_S

TK1_80_40

LpR_S

TNH

1_80_40

LpR_S

TNO

1_t80_40

LpR_S

TPO

1_80_40

mito

cp

rRNA

gene_sum

Number of reads mapping to plant genes from root libraries

0

5,000,000

10,000,000

15,000,000

20,000,000

25,000,000

30,000,000

LpR_FR

FULL1_80_40

LpR_FR

Ca1_80_40

LpR_FR

K1_80_40

LpR_FR

NH

1_80_40

LpR_FR

NO

1_80_40

LpR_FR

PO1_80_40

LpR_S

TFULL1_80_40

LpR_S

TCa1_80_40

LpR_S

TK1_80_40

LpR_S

TNH

1_80_40

LpR_S

TNO

1_t80_40

LpR_S

TPO

1_80_40

mito

cp

rRNA

gene_sum

Number of reads mapping to plant genes from root libraries

0

10,000,000

20,000,000

30,000,000

40,000,000

50,000,000

60,000,000

LpL_FRFULL1_80_40

LpL_FRCa1_80_40

LpL_FRK1_80_40

LpL_FRNH1_80_40

LpL_FRNO1_80_40

LpL_FRPO1_80_40

LpL_STFULL1_80_40

LpL_STCa1_80_40

LpL_STK1_80_40

LpL_STNH1_80_40

LpL_STNO1_80_40

LpL_STPO1_80_40

mito

cp

rRNA

gene_sum


0

10,000,000

20,000,000

30,000,000

40,000,000

50,000,000

60,000,000

LpL_FRFULL1_80_40

LpL_FRCa1_80_40

LpL_FRK1_80_40

LpL_FRNH1_80_40

LpL_FRNO1_80_40

LpL_FRPO1_80_40

LpL_STFULL1_80_40

LpL_STCa1_80_40

LpL_STK1_80_40

LpL_STNH1_80_40

LpL_STNO1_80_40

LpL_STPO1_80_40

mito

cp

rRNA

gene_sum


Of 918 endophyte-regulated plant genes 728 are differentially regulated in roots

(529 induced and 167 repressed) 14


Cluster 3: root-expressed genes induced

by endophytes, but expressed at lower level

in endophyte-free plants

Largest cluster of endophyte-regulated

plant genes

Annotation of endophyte-regulated plant

genes

Defence response genes

Chitin responsive genes

Innate immunity genes

Patterns of Expression in Endophyte-Regulated Plant Genes

Part of cluster 3 hierachical clusterPart of cluster 3 hierachical cluster

Symbiotum transcriptional

response to endophyte

presence is up-regulation of

defence-related genes in roots 15


Hierarchical clustering of C1Hierarchical clustering of C1

Cluster 1: root-expressed genes repressed by

endophytes

Annotation of endophyte-regulated plant genes

Transcription regulators

Max2 F-box LRR gene in

signalling of strigolactones


Clusters 10 and 11: shoot-expressed genes

repressed by endophyte

Annotation of 15 endophyte-regulated plant genes

3 MADS-box genes

2 blue light photoreceptors

1 cytokinin oxidase

Carbohydrate metabolism and transporters

16

17

Peramine

N-formylloline

Lolitrem B

Metabolome Analysis of Symbiota

Ergovaline

Metabolic Profiling of Natural Symbiota

Metabolic profiling across spectrum of genetic, geographic

and taxonomic diversity of endophytes from perennial ryegrass 17

18

Barsandra Tolosa Impact

Lolitrem B

0.00

0.50

1.00

1.50

2.00

2.50

3.00

NEA10 NEA11 NEA12 E1 STx x x

b

a

Ergovaline

0.00

0.50

1.00

1.50

2.00

2.50

3.00

NEA10 NEA11 NEA12 E1 STx x x

*

Janthitrem

0.00

0.50

1.00

1.50

2.00

2.50

NEA10 NEA11 NEA12 E1 STx

a

ab

b

x x

Peramine

0.00

0.50

1.00

1.50

2.00

NEA10 NEA11 NEA12 E1 ST

b a

a

*

* b

a

x x x

NEA10 NEA11 NEA12 E1 ST

Lo

litr

em

B

Erg

ova

lin

e

Ja

nth

itre

m

Pe

ram

ine

Metabolome Analysis of Symbiota Metabolic Profiling of Novel Symbiota

in Isogenic Hosts

Strong Gp x Ge effects on alkaloid toxin profiles

in defined symbiota with novel endophytes 18

Endophyte

strain

Putative

toxin profile

Endogenous

toxin profile

Isogenic

(confirmed)

toxin profile

Taxon

NEA10 Unknown -/E/n.d a -/E/P/- (Y) N. lolii

NEA11 E+P -/E/n.d a -/E/P/- (Y) Lp TG-2

NEA12 Unknown -/-/- -/-/-/J (Y) Lp TG-3

E1 Unknown n.d -/-/-/-

ST L/E/P L/E/P/- (Y) N. lolii

a Peramine not measured

Lp TG-3

19


X X X X

Adapted from Young et al, 2009

10 lolitrem biosynthetic genes

3 gene clusters

2 deletions (LtmE, LtmJ)

Pathway Analysis – Lolitrem Biosynthesis

19

20

Compound Bea02 Bro08 Imp04 San02 Tol03 Imp04 Bea02 Bro08 Imp04 San02 Tol03 Imp04 San02 Tol03 Bro08 Imp04 Bea02 Bro08 San02 Tol03

E- E- E- E- E- NEA10 NEA11 NEA11 NEA11 NEA11 NEA11 NEA12 NEA12 NEA12 E1 E1 ST ST ST ST

paspaline - - - - - + + + + + + + + + + + + + + +

13-desoxy paxilline - - - - - + + + + + + + + + - - + + + +

paxilline - - - - - + + + + + + + +(Trace) +(Trace) - - + + + +

terpendole I - - - - - + + + + + + + - + - - + + + +

prenylate terpendole I - - - - - + + + + + + + - + - - + + + +

terpendole C - - - - - + + + + + + + - + - - + + + +

lolitriol - - - - - - - - - - - - - - - - + + + +

lolitrem E - - - - - - - - - - - - - - - - + + + +

lolitrem B - - - - - - - - - - - - - - - - + + + +

lolitrem J - - - - - - - - - - - - - - - - - - - -

lolitrem K - - - - - - - - - - - - - - - - + + + +

paspalicine - - - - - + + + + + + + - + - - + + + +

paspalicinol - - - - - + + + + + + + + + - - + + + +

paspalininol - - - - - + + + + + + + - + - - + + + +

paspalinine - - - - - + + + + + + + - + - - + + + +

aflatrem - - - - - + + + + - + + - + - - + + - +

tryptophan + + + + + + + + + + + + + + + + + + + +

chanoclavine - - - - - + + + + + + - - - - - + + + +

secolysergine - - - - - - - - + + - - - - - - + + + +

agroclavine - - + + + + + + + + + + - - - + + + + +

setoclavine - - - + + + + + + + + - + + - + + + + +

elymoclavine - - - - - + + + + + + - - - - - + + + +

lysergic acid + + + + + + + + + + + + + + + + + + + +

lysergyl peptide lactam - - - - - + + - + - + - - - - - + + + +

lysergyl alanine - - - - - + + + + + + - - - - - + + + +

lysergamide - - - - - + + + + + + - - - - - + + + +

ergovaline - - - - - + +(Trace) + + +(Trace) + - - - - - +(Trace) + + +

lysergol - - - - - + + + + + + - - - - - + + + +

Peramine +(Trace) +(Trace) +(Trace) +(Trace) +(Trace) + + + + + + +(Trace) +(Trace) +(Trace) +(Trace) +(Trace) + + + +

Imidacloprid - - - - + - - - - + + - + + - - - - + -

4-Hydroxy-imidacloprid + - + + + + + + + + + + + + + + + + + +

Janthitrem I - - - - - - - - - - - + + + + +(Trace) - - - -

Janthitrem A - - - - - - - - - - - - - + + - + + + +

Janthitrem B - - - - - - - - - - - + + + + + - - - -

Janthitrem C + + + + + + + + + + + + + + + + + + + +

Peramine

Janthitrem

Host-EndophyteSymbiota

Lolitrems

Aflatrem

Ergot

Alkaloids

Metabolome Analysis of Symbiota Pathway Analyses – Lolitrems, Aflatrem, Ergot Alkaloids, Peramine and Janthitrems

20

21

Assessing Endophyte Stability and Symbiota Performance

Barsandra E- ST NEA11 NEA12

Bronsyn

A. Number of inoculations performed

ST NEA10 NEA11 NEA12 Total

Bea02 40 30 70

Bro08 80 75 155

Imp04 90 50 140

San02 80 50 130

Tol03 80 40 120

Total 0 370 0 245 615

B. Number of inoculations tested


Bea02 31 21 52

Bro08 59 50 109

Imp04 60 21 81

San02 64 31 95

Tol03 32 27 59

Total 246 150 396

C. Number of successful inoculations


Bea02 0 1 1

Bro08 1 0 1

Imp04 1 2 3

San02 0 1 1

Tol03 0 2 2

Total 2 6 8

D. Percent of successful inoculations


Bea02 0 1 1.0

Bro08 1.7 0 1.7

Imp04 1.7 9.5 11.2

San02 0 3.2 3.2

Tol03 0 7.4 7.4

Total 3.4 21.2 24.5

Stable association

Unstable association

Stable association

Unstable association

E- ST NEA11 NEA12

Phenome Analysis of Symbiota

21

22

Assessing Endophyte Effect on Symbiota Performance Shoot Fresh Weight in Response to Nitrate

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

E- NEA10 NEA11 NEA12 ST

Host-Endophyte Association

Fres

h W

eigh

t (g)

0.5 mM NO3-

2.5 mM NO3-

10.0 mM NO3-

Shoot fresh weightTiller Number in Response to Nitrate

0

10

20

30

40

50

60

70

80



Tille

r N

umbe

r

0.5 mM NO3-

2.5 mM NO3-

10.0 mM NO3-

Tiller number

Root fresh weightRoot Fresh Weight in Response to Nitrate

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00



Roo

t Fre

sh W

eigh

t (g)

0.5 mM NO3-

2.5 mM NO3-

10.0 mM NO3-

Root Dry Weight in Response to Nitrate

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70



Roo

t Dry

Wei

ght (

g)

0.5 mM NO3-

2.5 mM NO3-

10.0 mM NO3-

Root dry weight

Shoot Fresh Weight in Response to Nitrate

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00



Fres

h W

eigh

t (g)

0.5 mM NO3-

2.5 mM NO3-

10.0 mM NO3-

Shoot fresh weightTiller Number in Response to Nitrate

0

10

20

30

40

50

60

70

80



Tille

r N

umbe

r

0.5 mM NO3-

2.5 mM NO3-

10.0 mM NO3-

Tiller number

Root fresh weightRoot Fresh Weight in Response to Nitrate

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00



Roo

t Fre

sh W

eigh

t (g)

0.5 mM NO3-

2.5 mM NO3-

10.0 mM NO3-

Root Dry Weight in Response to Nitrate

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70



Roo

t Dry

Wei

ght (

g)

0.5 mM NO3-

2.5 mM NO3-

10.0 mM NO3-

Root dry weight

0.5 mM NO3-

2.5 mM NO3-

10.0 mM NO3-

Phenome Analysis of Symbiota

22

23

23 N-formylloline

Peramine

• Perennial ryegrass and • Tall fescue

Establish symbiota

with both:

NEA21 Morocco

NEA23 Tunisia

Novel endophytes for broad deployment discovered and characterised 23

Novel Fungal Endophytes for Forage Grasses Discovering Endophytes with Novel Bioactivity

and Broad Host Specificity

24

Forage Grass Microbiomes Meta-Transcriptomics of Ryegrass Microbiomes

Alphaproteobacteria, 654

Gammaproteobacteria,

493

Betaproteobacteria, 421

Actinobacteria, 341

Bacteroidetes, 294

Cyanobacteria/Chloroplast

, 235

Firmicutes, 136

Chlamydiae, 6

Spirochaetes, 12Chloroflexi, 15

Planctomycetes, 31

Deltaproteobacteria, 59

Deinococcus-Thermus, 23

Nitrospira, 4Tenericutes, 10

Epsilonproteobacteria, 7

Acidobacteria, 3

Fusobacteria, 2

Chrysiogenetes, 2

Deferribacteres, 2

Verrucomicrobia, 17

Diverse bacterial microbiomes revealed in forage grasses

Rapid and cost

effective RNA

profiling of

plant microbiomes

• Shoot and root microbiomes

• Meta-transcriptomics (16S rRNA)

• Over 2700 bacterial phyla in

perennial ryegrass microbiome

24

25

Forage Grass Microbiomes Shoot and Root Microbiomes in Perennial Ryegrass

Hierarchical clustering of bacterial counts classifies root treatments but not shoot treatments.

Meta-transcriptomics reveals differences in bacterial species

predominance in shoot and root microbiomes

Root microbiome profiles ‘descriptive’ of treatment (e.g. nutritional status) 25

26

Forage Grass Microbiomes Shoot and Root Microbiomes in Perennial Ryegrass

L_F

_FU

LL1

L_F

_FU

LL3

L_F

_FU

LL5

L_F

_Ca2

L_F

_Ca4

L_F

_K1

L_F

_K3

L_F

_K5

L_F

_NH

2L_

F_N

H4

L_F

_NO

1L_

F_N

O3

L_F

_NO

5L_

F_P

02*

L_F

_P04

*L_

S_F

ULL

1L_

S_F

ULL

3L_

S_F

ULL

5L_

S_C

a2L_

S_C

a4L_

S_K

1L_

S_K

3L_

S_K

5L_

S_N

H2

L_S

_NH

4L_

S_N

O1

L_S

_NO

3L_

S_N

O5

L_S

_P02

*L_

S_P

04*

R_F

_FU

LL1

R_F

_FU

LL3

R_F

_FU

LL5

R_F

_Ca2

R_F

_Ca4

R_F

_K1

R_F

_K3

R_F

_K5

R_F

_NH

2R

_F_N

H4

R_F

_NO

1R

_F_N

OL3

R_F

_NO

5R

_F_P

O2

R_F

_PO

4R

_S_F

ULL

1R

_S_F

ULL

3R

_S_F

ULL

5R

_S_C

a2R

_S_C

a4R

_S_K

1R

_S_K

3R

_S_K

5R

_S_N

H2

R_S

_NH

4R

_S_N

O1

R_S

_NO

3R

_S_N

O5

R_S

_PO

2R

_S_P

O4

Azospirillum sp

0

50

100

150

200

250

Azospirillum sp

Azospirillum sp

Azospirillum amazonense

Azospirillum sp

Azospirillum sp

Azospirillum amazonense

Azospirillum brasilense


Azospirillum lipoferum

Azospirillum sp

Azospirillum sp


Analysis of bacterial microbiome in symbiota reveals range of bacterial

species known to be N fixers and phytostimulators of grasses

Azospirillum species induced in number (under low N)

Associative nitrogen fixation

Synthesis of phytohormones

26

Integrative, Genomics-Assisted

F1 Hybrid Breeding of

Forage Grass Symbiota

Lessons and Prospects?

1. Breeding and Selection of Host Grass Only

Current Paradigm

2. Few Selective Recombinations in Long Breeding Cycle

4. Evaluation of Symbiota (i.e. Grass-Endophyte Associations)

3. Inoculation of Single Unselected Endophytes

5. Seed Generational Advance Limiting Heterosis

6. No Hybrid Varieties Limiting Value Capture

28

Lessons and Prospects?

1. Ab Initio Breeding and Selection of Symbiota

New Paradigm?

2. More Selective Recombinations in Shorter Breeding Cycle

4. More Accurate Evaluation of Symbiota

3. Exploit Broader Endophyte Diversity and Endophyte Effects

5. Exploit Heterosis and High-Impact Traits

6. Hybrid Varieties Enhancing Value Capture

29

Capture Ab Initio Plant Genotype X Endophyte Genotype Effects

Capture and Exploit Broader Endophyte Genotype Effects

on Symbiota Performance

Extend Concept of Synthetic Varieties to Both Partners of

the Symbiotum i.e. Grass Host and Endophyte

→ Deploy multiple endophyte and grass genotypes in populations

selected for optimal symbiota compatibility and performance

→ Breed and select ab initio symbiota for optimal symbiota compatibility

and performance rather than breed and select grass host only followed

by endophyte inoculation and symbiota evaluation

→ Exploit significant endophyte genotype effects on symbiota performance

well beyond pest resistance (and reduced animal toxicosis)

What Does This Mean?

30

Maximise Heterosis in Farmers’ Seed

Deliver F1 Hybrid Symbiota Varieties for Maximal On-Farm Impact

Reduce Generation Interval and Increase Selection Intensity

of Symbiota

→ Tailor genomic selection interventions in breeding cycle building on

simulated breeding schemes and sward-relevant phenotypes

→ Implement integrative, F1 hybrid symbiota breeding schemes building on

self-incompatibility allele typing

→ Produce F1 hybrid seed of symbiota deploying multiple endophytes and

high-impact traits

What Does This Mean?

31

Overcoming Bottle-Necks

• New tools for efficient, robust, low-cost, large-scale

generation of grass-endophyte symbiota

Method applicable to inoculation of 10s of endophytes in

100s of grass genotypes

Method applicable to inoculation of novel and designer

endophytes with de novo generated genetic variation

[i.e. induced mutagenesis (ionizing radiation, colchicine), genome editing, transgenesis]

Enabling tool for next-generation ab initio molecular breeding, selection

and evaluation of grass-endophyte symbiota [rather than breeding and selection of

grass host followed by endophyte inoculation and symbiota evaluation only]

High-Throughput, Large-Scale Endophyte Inoculation

32

Day 1 Day 3 Days 4-5 Days 6-7 Days 8-10 Day 1 Day 3 Days 4-5 Days 6-7 Days 8-10

Production of Artificial Seeds

Large-Scale Generation of Symbiota

Coating with single or multiple Ca-alginate matrix layers of ryegrass mature seed-derived embryos

Assessing germination frequency of artificial seeds

33

Inoculation of isolated

seed-derived embryos with endophyte mycelia followed by Ca-alginate coating into artificial seeds or double-coating of isolated seed-derived embryos with endophyte- containing inner Ca- alginate matrix

Coating seed-derived embryos with multiple endophytes into viable symbiota artificial seeds

a b c

First coating Second coating Endophyte outgrowth Germinating symbiota

Large-Scale Inoculation of Endophytes into Artificial Seeds Large-Scale Generation of Symbiota

Generating > 1,000 viable symbiota artificial seeds per FTE and day

Established symbiota plants with live endophytes in <50% artificial seeds. 34

Predicting Endophyte Stability in Stored Seed and Selecting

Stable Associations Using Accelerated Ageing

A method for Accelerated Ageing [i.e. 80% -100% RH for 4-7 days]

of seed (natural and artificial) with resident endophytes developed

The method allows to predict endophyte stability in stored

seed [range of endophytes assessed in single and different host genetic backgrounds]

The method allows to rank novel endophytes according to predicted

stability/viability in stored seed

[range of endophytes assessed in single host genetic background]

The method allows to select and rank symbiota according to their

stability


35

36

NEA12

0

20

40

60

80

100

120

Alto Bealey Bronsyn Trojan

Control

80% 4d

80% 7d

100% 4d

100% 7d

NEA10

0

20

40

60

80

100

120


Control

80% 4d

80% 7d

100% 4d

100% 7d

NEA11

0

20

40

60

80

100

120


Control

80% 4d

80% 7d

100% 4d

100% 7d

E1

E1

0

20

40

60

80

100

120

A l to Beal ey Br onsyn T r oj an Endo

Cont r ol

80% 4d

80% 7d

100% 4d

100% 7d

E1

0

20

40

60

80

100

120

A l to Beal ey Br onsyn T r oj an Endo

Cont r ol

80% 4d

80% 7d

100% 4d

100% 7d

Accelerated ageing [i.e. 100%RH for 4d or 7d] allows ranking endophytes for

compatibility and selecting for endophyte genotype-host genotype stability

Using Accelerated Ageing to Select for Symbiota Viability and Stability Assessed endophyte viability and stability of symbiota after accelerated ageing treatment of seed

Selection for Symbiota Stability

36

37

Rapid Early Assay of Endophyte Viability in Symbiota

37


A fast, reliable and low-cost method for determining endophyte

viability in perennial ryegrass seeds, seedlings and established

symbiota

Assay Requirements:

1. Rapid determination: 3-5 day old epicotyls

2. Robust and reliable

3. Sensitive for use in single seed to seed batches

4. Specific to Neotyphodium endophytes

(i.e. does not detect other fungi)

5. Detects live endophyte only

Seed with endophyte

38 38

Assaying Endophyte Viability Metabolomics-Based Assay

Assays developed based on:

• Genotyping

• Early gene expression

• Production of indicator metabolites

Seed

germination

Harvest

epicotyls

Dark

Light

Day 1 Day 4 Day 5 Day 6

Metabolite

extraction

Direct

Injection MS

Set-up

Data

analysis

With Endophyte

Without Endophyte

Detection of E- seed

Rapid (≤6 days), low cost (<$1/sample) assay – 5X cheaper and 5X faster

39 39

Increasing Accuracy and Reducing Cost of Phenotyping

Low-cost, high-throughput, accurate methods for large-scale

phenotyping of individual plants for herbage quality traits

Robust, reliable methods enabled by automated workflows


Low-cost, high-throughput, accurate methods for large-scale,

multisite phenotyping of key traits at sward level

Field-based phenomics (from individual plant to farmer’s paddock)

Laboratory-based molecular phenomics

Generating low-cost, high-throughput, accurate, relevant

phenotypes for genomics-assisted molecular breeding

40

Rainout Shelters Precise water-stress treatments

Automated Assessments Vegetative biomass Quality traits – CP/WSC/ME/Minerals Persistence traits – Biomass

over time Stress related traits

Active Optical Sensors Canopy greenness &

photosynthetic capacity Normalised difference

vegetative index output Forage quality

Field-Based Phenomics

40

41

Non-Destructive Forage Yield Estimation Using Normalized

Difference Vegetation Index


GreenSeeker Aphex hexacopter

41

42

peramine

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101105109113117121125129133137141145149153157161165169173177

Sample ID

Am

ount

of

Per

amin

e

lolitrem B

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

1 8 15 22 29 36 43 50 57 64 71 78 85 92 99 106 113 120 127 134 141 148 155 162 169 176

Sample ID

amou

nt o

f lo

litre

m B

ergovaline

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

1 8 15 22 29 36 43 50 57 64 71 78 85 92 99 106 113 120 127 134 141 148 155 162 169 176

Sample ID

amo

un

t of e

rgo

valin

e

peramine

ergovaline

lolitrem B

Exploiting genotype x genotype

interactions

Overcoming limitations of current

paradigm: breeding hosts and

evaluating symbiota

Setting the basis for molecular

breeding of symbiota

Proof of concept in semi-

quantitative toxin profiling of

symbiota breeding population

(i.e. 80 Bealey NEA2/NEA6)

Significant variation in alkaloid

profile and content

Molecular Phenomics of Symbiota Molecular Phenotyping to

Enable Symbiota Selection

42

43

Molecular Phenomics of Symbiota

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127 133 139 145 151 157 163 169 175

lolitrem B

ergovaline

peramine

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

lolitrem B

ergovaline

peramine

Top 20 peramine producing symbiota

Molecular Phenotyping to Enable Symbiota Selection

Selection of symbiota within breeding population with favourable toxin profiles

• high peramine, low ergovaline, no lolitrem B

Molecular breeding of symbiota capturing ab initio Gp x Ge effects 43

44 44

Refined breeding schemes

Phenotyping tools at acceptable cost

Genotyping tools at acceptable costs

Computational tools to handle data

and empowering breeders

Genomic Selection Selection candidates

Genotypes

Selected parents

Estimated

breeding values

Prediction equation

Genomic Breeding Value = w 1 x 1 +w 2 x 2 +w 3 x 3 ……..

Reference population

Genotypes

Phenotypes

Increasing Rate of Genetic Gain via Genomic Selection


45 45

Rates of Selective Breeding, Genetic Gain and Improvement

M1 B M2

(A) (B)

(C)

(D)

(E)

Genomic selection

Update

prediction

equation

Multi-site environment trials

F1 Production

Seed production

(F2 Production)

Selection under grazing and/or

visual assessment

Varietal construction

Seed production

Multi-environment

plot trials

1 varietal release

Base population

establishment

c. 1,000 – 10,000

Individuals

c. 100,000 Individuals

Reduction in

individuals by a

factor of 10

Selective

Recombination

Non-Selective

Recombination

Selective

Recombination

c. 1-10 Varieties

Multi-environment

plot trials

Less than 100

varietiesNon-Selective

Recombination

Less than 100 varieties

F1 Production

Seed production

(F2 Production)


visual assessment


Seed production

Multi-environment

plot trials

1 varietal release

Base population

establishment

c. 1,000 – 10,000

Individuals


Reduction in

individuals by a

factor of 10

Selective

Recombination

Non-Selective

Recombination

Selective

Recombination

c. 1-10 Varieties

Multi-environment

plot trials

Less than 100


Recombination


F1 Production

Seed production

(F2 Production)


visual assessment


Seed production

Multi-environment

plot trials

1 varietal release

Base population

establishment

c. 1,000 – 10,000

Individuals


Reduction in

individuals by a

factor of 10

Selective

Recombination

Non-Selective

Recombination

Selective

Recombination

c. 1-10 Varieties

Multi-environment

plot trials

Less than 100


Recombination


2 selective recombination steps

– 10 years

2 selective recombination steps – 3 years

Genomic Selection

Computational simulation of commercial ryegrass breeding program to

optimise application of genomic selection

Genomic-estimated breeding values for key traits in ryegrass breeding

46 46

Exploiting Heterosis via Novel Hybrid Breeding Scheme

Candidate genes for Self-Incompatibility loci (S and Z) discovered and

functionally characterised

An F1 hybrid breeding scheme designed and being piloted


Fertilisation

S1Z1

S1Z2 S2Z2 S1Z3S3Z1

S3Z3

S1S2Z1Z2

S1S2Z1Z2

S1Z1

S1Z2

S2Z1

S2Z2

PistilPollen

(haploid)(diploid)

Pistil

Anther

A method for SI allele prediction developed

47 47

Os0

4g06

4510

0 (T

C1018

21)

Os0

4g06

4520

0

Os0

4g06

4550

0

Os0

4g06

4560

0

Os0

4g06

4570

0

Os0

4g06

4590

0

Os0

4g06

4610

0

Os0

4g06

4650

0

Os0

4g06

4670

0

Os0

4g06

4680

0

Os0

4g06

4690

0

Os0

4g06

4710

0

Os0

4g06

4720

0

Os0

4g06

4730

0 (T

C1169

08)

Os0

4g06

4780

0 (T

C8905

7)

Os0

4g06

4790

0

Os0

4g06

4800

0

Os0

4g06

4820

0

Os0

4g06

4840

0

Os0

4g06

4850

0

Os0

4g06

4860

0

Os0

4g06

4870

0

Os0

4g06

4880

0

Os0

4g06

4890

0

Os0

4g06

4910

0

Os0

4g06

4920

0

Os0

4g06

4950

0

Os0

4g06

4960

0

Os0

4g06

4970

0

Os0

4g06

4990

0

Os0

4g06

5000

0 (b

cd26

6)

BAC8-E18BAC 119-E12

BAC50-H02

BAC118-B23

BAC87-P20

BAC67-H10 BAC85-A01 BAC27-A19 BAC93-M20 BAC90-J24

LpT

C101821

LpV

Q

LpO

s04g0645500

LpO

s04g0645600

LpT

C116908

LpT

C89057

LpO

s04g0648400

LpO

s04g0648500

LpO

s04g0648600

LpO

s04g0648700

LpO

s04g0648800

LpO

s04g0648900

LpO

s04g0649200

LpO

s04g0649100

Lpbcd266

BAC127-K20

BAC65-A01BAC79-L12

LpO

s06g0607900

LpD

UF

247

LpO

s03g0193400

LpO

s06g0607800

LpO

s11g0242400

LpO

s10g0419600

LpO

s06g0607900

LpO

s04g0274400

Rice chr.4

Brachypodium Bd5

Perennial ryegrass

Z locus region

Conserved genes

Specific genes

Os0

4g06

4510

0 (T

C1018

21)

Os0

4g06

4520

0

Os0

4g06

4550

0

Os0

4g06

4560

0

Os0

4g06

4570

0

Os0

4g06

4590

0

Os0

4g06

4610

0

Os0

4g06

4650

0

Os0

4g06

4670

0

Os0

4g06

4680

0

Os0

4g06

4690

0

Os0

4g06

4710

0

Os0

4g06

4720

0

Os0

4g06

4730

0 (T

C1169

08)

Os0

4g06

4780

0 (T

C8905

7)

Os0

4g06

4790

0

Os0

4g06

4800

0

Os0

4g06

4820

0

Os0

4g06

4840

0

Os0

4g06

4850

0

Os0

4g06

4860

0

Os0

4g06

4870

0

Os0

4g06

4880

0

Os0

4g06

4890

0

Os0

4g06

4910

0

Os0

4g06

4920

0

Os0

4g06

4950

0

Os0

4g06

4960

0

Os0

4g06

4970

0

Os0

4g06

4990

0

Os0

4g06

5000

0 (b

cd26

6)

BAC8-E18BAC 119-E12

BAC50-H02

BAC118-B23

BAC87-P20

BAC67-H10 BAC85-A01 BAC27-A19 BAC93-M20 BAC90-J24

LpT

C101821

LpV

Q

LpO

s04g0645500

LpO

s04g0645600

LpT

C116908

LpT

C89057

LpO

s04g0648400

LpO

s04g0648500

LpO

s04g0648600

LpO

s04g0648700

LpO

s04g0648800

LpO

s04g0648900

LpO

s04g0649200

LpO

s04g0649100

Lpbcd266

BAC127-K20

BAC65-A01BAC79-L12

LpO

s06g0607900

LpD

UF

247

LpO

s03g0193400

LpO

s06g0607800

LpO

s11g0242400

LpO

s10g0419600

LpO

s06g0607900

LpO

s04g0274400

Rice chr.4

Brachypodium Bd5

Perennial ryegrass

Z locus region

Conserved genes

Specific genes

F1 Hybrid Breeding and SI Allele Prediction

48

Advances in Forage Systems Biology

Summary

Genome, Transcriptome, Proteome, Metabolome and Phenome

Forage Symbiomes and Microbiomes – Exploiting Supplementary

Genomes

Lessons from Systems Biology of Forage Symbiomes

Integrative, Genomics-Assisted Hybrid Breeding of Symbiota

Prospects for Trebling Genetic Gain

49

Acknowledgements P. Badenhorst, N. Cogan, H. Daetwyler, S. Davidson, P. Ekanayake, S. Felitti, J. Forster, K. Fulgueras, K. Guthridge, M. Hand, B. Hayes, M. Hayden, I. Hettiarachchi, D. Isenegger, J. Kaur, G. Latipbayeva, T. Le, Z. Lin, Z. Liu, C. Ludeman, E. Ludlow, R. Mann, L. Pembleton, M. Rabinovich, M. Ramsperger, P. Rigault, S. Rochfort, T. Sawbridge, K. Shields, L. Schultz, H. Shinozuka, K. Smith, G.Tao, P. Tian, P.X. Tian, J. Tibbits, Y. Ran, E. van Zijll de Jong, J. Wang, T. Webster

C. Inch, S. van der Heijden, M. Willocks

Cluster 1 Cluster 3Cluster 2 Cluster 4

Cluster 5 Cluster 7 Cluster 8Cluster 6

Cluster 9 Cluster 10 Cluster 11 Heat map depiction of average cluster

expression.

Columns are

Cluster Number, Cluster popn., Cluster

Diversity .



Cluster 9 Cluster 10 Cluster 11



Cluster 9 Cluster 10 Cluster 11 Heat map depiction of average cluster

expression.

Columns are


Diversity .

Heat map depiction of average cluster

expression.

Columns are


Diversity .

Samples in order

Leaf Free Root Free Leaf ST Root ST

Replete Ca K NH4 NO3 PO4 Replete Ca K NH4 NO3 PO4 Replete Ca K NH4 NO3 PO4 Replete Ca K NH4 NO3 PO4


11 clusters generated by Self Organizing Trees Algorithm analysis of endophyte-regulated plant genes

Cluster 1: root-expressed genes repressed by endophytes

Clusters 3 and 4: root-expressed genes induced by endophytes


51


Cluster 2: root-expressed genes

repressed by endophytes as well as

root-expressed genes induced by

endophytes

Differential gene expression

driven by NH4 responsiveness


Hierarchical clustering of genes in cluster 2Hierarchical clustering of genes in cluster 2

Endophyte-regulated

plant genes differentially

regulated in roots

depending on nutritional

symbiota status

52

53

1. Alkaloid (LEPJ) profiles of symbiota

(i.e. E+) versus E- isogenic host plants

2. Alkaloid profiles of symbiota with diverse

endophyte panel in a single isogenic host

3. Alkaloid (LEPJ) profiles of symbiota with

endophytes from different taxonomic

groups across same host panel

Metabolic Profiling of Novel Symbiota in Isogenic Hosts Detailed characterisation of known

alkaloids and their precursors


Analysis of Gp x Ge effects on symbiota stability and toxin profile 53

We MUST We NEED

2X Productivity Growth 3X Genetic

Gain

55 55

Field-Based Phenomics Low-Cost, High-Throughput, Field-Based Phenotyping: Pheno-Lab

Method Consumables Assets Labour Total Cost Time/sample (min)

NIR 0 0.61 10.75 11.36 15

HPLC 7.23 7.08 0.56 14.86 40.47

Enzymatic Assay 1.37 0.27 0.67 2.31 1.17

MALDI-TOF 1.79 0.42 1.05 3.26 1.48

In-field NIR and yield 0 0.61 1.43 2.04 2

Costs ($) per sample (i.e. plant)

Accurate, low-cost, high-throughput phenotyping of forages

56

0

0.1

0.2

0.3

0.4

0.5

0.6

cont

rol 1 2 3 * 4 5 6 7 8 9 10 11 12 13 14 15 16 * 1

7 18

NEA12 colonies treated (no.)

Size

of m

ycel

ia (c

m, L

ogN

)

* **

Analysis of growth rate in culture

after 8 weeks

Initial Screen: Analysis of variance identified two

colonies significantly different to the control

NEA12v17 grows significantly faster (p<0.01**)

NEA12v4 grows significantly slower (p<0.05*)

Validation Screen: Student’s t-tests identified

two colonies significantly different to the control

NEA12v17 grows significantly faster (p<0.01**)

NEA12v15 grows significantly slower (p<0.01**)

Analysis of growth rate in culture

over 5 weeks

In Vitro Growth of NEA12 Variant Strains

Phenome Analysis of Variant Endophytes

Altered phenotypes (e.g. growth rates) observed in variant endophytes 56

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5

Week

Gro

wth

(m

m)

NEA12

NEA12v4

NEA12v5

NEA12v6

NEA12v13

NEA12v14

NEA12v15

NEA12v17

57 Gene present Gene absent Gene partially present


Identification of core and flexible genomes in Neotyphodium endophytes 57

58

easH easA

Intergenic deletions in eas gene cluster in AR1 endophyte

AR1

(Ergo-)

ST

(Ergo+)

CONTIG_29770

lpsB easG easF easE

CONTIG_29770


Intergenic deletions and SNP causing truncated lpsA also lead to inability to produce ergovaline 58

59

Phenome Analysis of Variant Endophytes Antifungal Bioassays of NEA12 Variant Strains Drechslera brizae (11 dpi) Phoma sorghina (11 dpi)

NEA12 v14

NEA12 v13

NEA12 v6

NEA12 v5

NEA12

Rhizoctonia cerealis (9 dpi)

NEA12 v14

NEA12 v13

NEA12 v6

NEA12 v5

NEA12

NEA12 v14

NEA12 v13

NEA12 v6

NEA12 v5

NEA12

Altered phenotypes (e.g. bioactivities) observed in variant endophytes 59

F1 Hybrid Breeding Designs

Endophyte Trait Diversity

Some Key Considerations

Endophyte Deployment

Self-Incompatibility

Value Modelling and Impact Delivery

Accurate, Low-Cost Genotypes and Phenotypes

60

Selection for Symbiota Stability

61

Germination of seeds after AA

treatment and storage

Growth of germinated seedlings

in soil for eight weeks

Assessment of endophyte status by

ELISA

Accelerated ageing treatment

• Optimised conditions identified (e.g. 80% humidity, 4-7 days)

• Variation identified between endophytes and grass cultivar combinations

Predicting Endophyte Stability in Stored Seed and Selecting

Stable Associations Using Accelerated Ageing

62

Experimental Work Flow for Colchicine Mutagenesis

n nucleus

n and 2n?

Colchicine treatment

(0-0.2% w/v)

3 weeks, 22oC, 150rpm, dark

Protoplast

preparation

4 weeks, 22oC, dark

Colony

subculture

Analyse for change in nuclei

size via flow cytometry

Stained cells

Colony

regeneration

A

C D

B B A

C

D

Protoplast

preparation

(single colonies)

4 weeks, 22oC, dark

SYBR Green I

staining of nuclei

Generation of Novel N. lolii Genotypes

De Novo Generation of Variant Endophytes

62

63

Experimental Work Flow for X-Ray Mutagenesis

Detection of target gene mutants using high through-put multiplex PCR

analysis for target gene presence and absence and by genome survey

sequencing

Single colonies isolated

Mutant detection

Protoplast preparation

Potato dextrose broth for 4 - 14 days

Recovery period (10 - 14 days) Repeated radiation

Exposure to ionising radiation caesium source

(10 - 30 Gy ) - )

Recovery: 4 - 6 weeks, 22 o C, dark - o

B A B A A

- -

15 days, 22oC, dark

Generation of Novel N. lolii Genotypes


X-ray mutagenesis for generation of variant endophytes 63

64


Generation of Fluorescently Marked Endophytes

ST:sgfpE1:DsRed NEA12:sgfp

e

e

e

e

e

e

Reporter Endophytes to Develop Endophyte Hybridisation

Methodologies and Study Host Colonisation

Agrobacterium-mediated transformation of N. lolii

and LpTG-3 endophytes with fluorescent reporter genes 64

65


65

Proof-of-Concept for Enhancing Bio-protective Properties

Agrobacterium-mediated transformation of janthitrem-producing

endophyte for perA expression and peramine production

248.15022

N H

O

O

H O O

O

O

H O

H

C h e m i c a l F o r m u l a : C 3 9 H 5 1 N O 7

E x a c t M a s s : 6 4 5 . 3 6 6 5 5

N

N

O

N

N H 2

H 2 N

C h e m i c a l F o r m u l a : C 1 2 H 1 7 N 5 O

E x a c t M a s s : 2 4 7 . 1 4 3 3 1

Janthitrem I

peramine

[M+H]

[M+H] 646.37238

pEND0025 20395 bp

25bp RB

25bp LB

attB1

attB2

SpecR/ StrepR

pBR322 origin

PVSI origin

PVSI STA region

hph

PerA gene

p gpd

P trpC

T trpC

T trpC

Peramine Biosynthesis perA Gene Expression Vector

Generation of Transgenic LpTG-3 Endophytes for Peramine Production

N. lolii ST LpTG-2 NEA11 LpTG-3 NEA12 P P J

201bp

PerA gene

414bp

Selectable marker gene

66

HTP method as NIR reference Environmental and meteorological data gathering of each trial site

Harvesting of plant material

No Sample Preparation e.g. oven drying and grinding

In-Field Biomass

In-Field Forage Quality

Forage Mobile Pheno-Lab Digital Image Library


66

67

0.00

100.00

200.00

300.00

400.00

500.00

600.00

EB

AS

E

EB

AS

E1M

EB

AS

E1M

-Pp

eak

EB

AS

E1M

+P

pea

k

EB

AS

E+

3.0

4%

DM

Y_

Milk

EB

AS

E+

9.9

2%

DM

Y_

SR

EB

AS

E1C

EB

AS

E1S

R

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no

mic

va

lue

(A

U$

pe

r h

ecta

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ela

tive

to

BA

SE

sce

na

rio

Scenario

Economic Value of Forage Traits (Elliott, relative to base scenario)

Modelling Value and Delivering Impact

67

advances and prospects in forage systems biology and molecular...

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